The Maize rough sheath2 Gene and Leaf Development Programs in Monocot and Dicot Plants

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Science  02 Apr 1999:
Vol. 284, Issue 5411, pp. 154-156
DOI: 10.1126/science.284.5411.154


Leaves of higher plants develop in a sequential manner from the shoot apical meristem. Previously it was determined that perturbed leaf development in maize rough sheath2 (rs2) mutant plants results from ectopic expression ofknotted1-like (knox) homeobox genes. Here, thers2 gene sequence was found to be similar to theAntirrhinum PHANTASTICA (PHAN) gene sequence, which encodes a Myb-like transcription factor. RS2 and PHAN are both required to prevent the accumulation of knox gene products in maize and Antirrhinum leaves, respectively. However,rs2 and phan mutant phenotypes differ, highlighting fundamental differences in monocot and dicot leaf development programs.

In both monocot and dicot plants, leaf initiation requires the early specification of founder cells in the meristem (1). The acquisition of founder cell identity appears to be determined by a class of homeobox genes known asknox that are normally expressed in the meristem but not in the founder cells (2, 3). Ectopic expression of the maize knotted1 (kn1) gene (and related dicot genes) often leads to the organization of new meristems in dicot leaves but not in monocot leaves (4). Thus, inappropriateknox gene expression can modify developmental fate within certain limits. Loss-of-function mutations in the maize kn1gene result in defects in shoot meristem maintenance (5). The phenotypes of recessive rs2 mutants resemble those induced by dominant mutations in rough sheath1 (rs1) and other kn1-like genes [(6) and Web Fig. 1(7)]. These include vascular tissue aberrations, ligular displacement toward the leaf tip, and the presence of knotlike outgrowths of aberrantly differentiated tissues. Because rs2mutants ectopically express mRNA encoded by the homeobox geneskn1, rs1, and liguleless3(lg3) (6), the rs2 gene may be a general repressor of knox gene expression during leaf development. To understand the mechanism of rs2 gene action, we cloned the rs2 gene using the transposable element Spm as a molecular tag [Fig. 1A and Web Fig. 2(7)]. Sequence analysis revealed that thers2-twd allele contains an Spm element inserted 21 base pairs (bp) after the predicted translation start site. Anotherrs2 allele (rs2-2.37) generated by transposon Mutator (Mu)–directed tagging was found to harbor a 1.3-kb insertion in the putative rs2gene [Web Fig. 2 (7)]. Further Southern (DNA) analysis of the original rs2 reference allele (referred to hereafter as rs2-R), with a probe immediately adjacent (3′) to the Spm insertion in rs2-twd, demonstrated that at least some of the cloned gene is deleted in thers2-R allele (Fig. 1B). Because polymorphisms were identified that segregated with all three rs2 mutant alleles, these data confirmed that we had cloned the rs2 gene.

Figure 1

Cloning of the rs2 gene. (A) Hybridization of DNA flanking the Spm element in rs2-twd to genomic DNA isolated from a line segregating rs2-twd mutants. Lane 1,rs2-twd/rs2-twd; lane 2, +/rs2-twd; lane 3, +/+. Genomic DNA was digested with Bam H1, and the filter was hybridized with the putative rs2 gene fragment. Thers2 fragment was isolated from a λZAP clone of a 6.3-kbSpm-hybridizing fragment [Web Fig. 2 (7)]. Flanking sequences 3′ to Spm were isolated. The putativers2 gene fragment detects a polymorphism that segregates with the rs2-twd mutation. The mutant allele is detected as a 6.3-kb fragment, and the wild-type allele is detected as an 11.0-kb fragment. (B) Genomic Southern blot of DNA from a line segregating the rs2-R allele. Genomic DNA was digested with Hind III, and the filter was probed with the same fragment as in (A). Lane 1, rs2-R/rs2-R; lane 2, +/rs2-Ror +/+. The absence of a hybridizing fragment in homozygousrs2-R plants suggests that the rs2-R allele represents a deletion. The wild-type allele is represented by a 1.8-kb fragment.

Figure 2

The rs2 gene sequence. (A) Gap alignment of the predicted PHAN (GenBank accession number AJ005586) and RS2 (GenBank accession numberAF126489) polypeptides. (B) Schematic summary of thers2 transcription unit. The positions of the Spminsertion in rs2-twd (s) and of the Mu insertion in rs2-2.37 (m) are marked. Open boxes depict the transcribed region, diagonal hatch the translated region, black bar the predicted myb domain, and gray bar the minimum length of the deletion in the rs2-R allele.

Sequence analysis revealed that the rs2 gene encodes a protein that shares sequence similarity (62.9% identity, 76.7% similarity) with the Myb-like protein encoded by the PHANTASTICA(PHAN) gene of Antirrhinum (Fig. 2A) (8). On the basis of reverse transcription–polymerase chain reaction (RT-PCR) experiments (9) and hybridization to genomic DNA (Fig. 1B) we predict that the deletion in the rs2-R allele spans most of themyb domain (Fig. 2B). The Spm insertion in the rs2-twd allele is also in the myb domain (Fig. 2B). Analysis by 5′ rapid amplification of cDNA ends (RACE) and sequencing of a genomic clone spanning the locus revealed the presence of an intron in the 5′ untranslated region of the rs2 gene (Fig. 2B). This feature is also seen in the Antirrhinum PHANgene (8).

In rs2-R and rs2-twd mutants, KNOX proteins accumulate ectopically in leaf tissue (Fig. 3A) (6). Similarly, RT-PCR demonstrated that at least one knox-like gene,AmSTM1, is ectopically expressed in Antirrhinum phan mutant leaves (Fig. 3B). These data suggested that RS2 and PHAN suppress the accumulation of knox gene products in the leaves of wild-type plants and are thus likely to be expressed in leaf tissue. In situ hybridization confirmed this prediction (Fig. 3, C, D, and E) (8). We detected rs2 transcripts throughout the plastochron 1 (P1) leaf. (Plastochron denotes the interval between initiation of leaves such that the primordium closest to the meristem is P1, the next one out from the meristem is P2, and so on). This expression pattern is consistent with the fact thatknox genes are ectopically expressed in rs2mutants from P1 onward (6). From P2 through P5,rs2 transcripts gradually become restricted to the vascular tissue and the leaf margins. After P5, further restriction confines transcripts to the vascular tissue. We did not detect rs2transcripts in the shoot apex of rs2-R mutants (Fig. 3F). Consistent with the idea that RS2 negatively regulates knoxgene expression, rs2 and knox genes are expressed in mutually exclusive domains (Fig. 4). Transcripts of rs2 were detected in leaf tissue but not meristematic tissue, whereas knox-like rs1transcripts were absent from leaf tissue but present in meristematic tissue.

Figure 3

Localization of rs2,knox, and AmSTM1 gene products in shoot apices. (A) Immunolocalization of KNOX protein in ars2-twd mutant apex, using an antibody raised to RS1. Note the ectopic accumulation of KNOX proteins around the leaf vasculature. (B) RT-PCR of AmSTM1 in phan mutant (lane 1) and wild-type (lane 2) Antirrhinumtotal leaf RNA. On the basis of partial genomic sequence data (8,10), primers were designed to position −24 to −1 relative to the final intron and to a 21-bp sequence in the 3′ untranslated region. The predicted PCR product from cDNA is 368 bp. PCR products were resolved on a 1% agarose gel that was blotted and probed sequentially with AmSTM1 fragments (8) and maize ubiquitin (ubi) fragments (3). Lane 3 is a “no template” control. (C) In situ localization ofrs2 transcripts in a transverse section of a wild-type maize apex. (D) Similar to (C) but at a higher magnification. (E) In situ localization of rs2 transcripts in a section close to the median of a wild-type maize apex. (F) In situ localization of rs2 transcripts in a transverse section of a rs2-R mutant apex. Scale bars = 100 μm. Plastochron (P) number of leaf primordia is indicated. White arrows denote the position of vascular bundles. Asterisks are placed near the center of the meristem in each case.

Figure 4

Transcript accumulation patterns ofrs2 and rs1 genes in wild-type maize tissues. The mRNA (1.5 μg) was analyzed by RNA gel blot analysis. Lane 1, leaf sheath; lane 2, leaf blade; lane 3, immature ear; lane 4, immature tassel. The blot was probed with the rs2 fragment as in Fig. 1A and with rs1 and ubiquitin (ubi) fragments as in (5). The rs2 transcript is 1.6 kb.

Our data indicate that RS2 regulates knox homeobox gene expression patterns during leaf development. However, RS2-mediated repression of knox gene expression is independent from the initial down-regulation required for the establishment of the leaf founder-cell population at P0 (6). Thus, RS2 is mainly involved in the maintenance of leaf cell fate as opposed to early initiation events. The nonuniformity of ectopic knoxgene expression in rs2 mutants (Fig. 3A) (6) may indicate that RS2 is only one component of a broader mechanism required to correctly compartmentalizeknox gene expression in the shoot body.

The rs2 and PHAN gene products are more similar to one another (62.9% identity) than to any other myb gene product. In addition, rs2 is more similar to PHANthan to a second PHAN-like gene in Antirrhinum(49.5% identity) (10). The sequence conservation (Fig. 2A), similarity in expression patterns (Fig. 3, C, D, and E) (8), and ectopic accumulation of knox gene products in mutant leaves (Fig. 3, A and B) suggest that rs2 andPHAN are functional orthologs. If RS2 delimits the expression of knox genes, all aspects of the rs2mutant leaf phenotype can be explained on the basis of ectopic accumulation of KNOX proteins in leaves. That is, the mutant phenotype resembles that seen in dominant Knox maize mutants. At least one knox-like gene (AmSTM1) is also ectopically expressed in phan mutant leaves (Fig. 3B). This concurs with the finding that expression domains of PHAN andAmSTM1 are mutually exclusive during wild-typeAntirrhinum development (8). The radially symmetrical phenotype of phan mutant leaves (11) (which is not seen in rs2 mutant leaves) could be explained if the perturbed expression of STM-like genes results in developmental retardation (12) and the failure to interpret signals that instruct the young leaf primordium to grow laterally and produce a lamina. Because leaf primordia are less flattened at P1 inAntirrhinum than in maize, an early developmental block could restrict the elaboration of dorsiventrality inAntirrhinum but would be less likely to do so in maize.

Morphologists believe that maize leaves are derived from different regions of the primordium than dicot leaves. Maize, like many other monocots, elaborates leaves entirely from the lower leaf zone of the primordium, the “unterblatt” (13). In contrast,Antirrhinum (and other dicots) elaborate leaves (petioles and lamina) from an upper leaf zone, the lower zone functioning only to ensheath the stem and to generate stipules when stipules are present (13, 14). When knox genes are ectopically expressed in maize leaves, the one phenotype common to all fiveknox genes tested is that the distal region of the leaf, the blade, is transformed to a proximal cell identity (sheath or auricle, depending on the timing of ectopic expression) (12,15). If ectopic knox gene expression acted similarly inAntirrhinum, one would expect to be able to see equivalent transformations: the more distal lamina would be transformed into petiole (or perhaps even into stemlike tissue). The petiole is a largely unifacial region; the abaxial (ventral) surface of the petiole dominates, and the base of the petiole is almost all ventral and symmetrical. Thus, a petiolization of the lamina could convey the impression of loss of dorsiventrality. In our view, the morphology ofphan leaves (11) is consistent with the conclusion that phan mutants “petiolize” the leaf. Thus,rs2 and PHAN appear to be functioning in similar gene regulatory networks to condition similar leaf regional transformations, with outcomes diverging because of differences in how the monocot maize and the dicot Antirrhinum elaborate laminae.

  • * Present address: CERES Incorporated, 3007 Malibu Canyon, Malibu, CA 90265, USA.

  • To whom correspondence should be addressed. E-mail: jane.langdale{at}


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